Substrate and mechanistic investigation of the tannases CbTan1 and 2 - serine hydrolases active on gallotannins

Conference poster, 2022

Tannases (tannin acyl hydrolases, EC 3.1.1.20) are serine-based α/β-hydrolases that can degrade gallotannins, which are secondary metabolites that inhibit microbial growth by binding to and precipitating proteins, and that are especially enriched in tree bark. Gallotannins are diverse, as they can contain carbohydrate, aliphatic or aromatic groups. Tannases can help microbes avoid the toxic effect of tannins, or even enable their metabolism.

Only a few tannases have been characterized structurally or biochemically, and these can be phylogenetically grouped based on the presence or absence of an acidic residue in the canonical Ser-His-Asp catalytic triad. Recently we produced and characterized three tannases from the anaerobe Clostridium butyricum (CbTan1-3). CbTan1 contains an amidic residue (Gln) in place of a catalytic acidic residue, while CbTan2 and 3 contain a catalytic aspartic acid, making these useful enzymes for investigating the mechanistic differences between types of tannases.

Based on crystal structures and AlphaFold2 models, we generated mutant tannases to probe both the substrate binding and mechanism of this understudied class of hydrolases. We show that larger substrates, such as epigallocatechin gallate (EGCG), glucogallin, and aliphatic gallotannins, can be accommodated by modifying or entirely removing the tannase cap domains. Attempts to “restore” the catalytic acid in CbTan1 resulted in reduction of activity, suggesting a complex role for the amidic residue. Conversely, CbTan2 catalysis was dramatically decreased when the catalytic aspartic acid was replaced with an asparagine.